Immunogenicity of liposomal model membranes sensitized with spin-labeled haptens and topographical distribution of haptens on the membranes

The relation between the in vitro immunogenicity of phosphatidylcholine liposomes containing 2,4-dinitrophenyl-6-N-aminocaproylphosphatidylethanolamine (DNP-Cap-PE) as a hapten and the topographical distribution of the haptens on lipid membranes was studied. In distearoylphosphatidylcholine liposomes, the immunogenicity increased with increase of cholesterol content in the liposomal membranes. The electron spin resonance spectra of spin-labeled DNP-Cap-PE in distearoylphosphatidylcholine liposomes indicated that cholesterol affected the topographical distribution of spin-labeled DNP-Cap-PE on the membranes. In the absence of cholesterol, a considerable amount of haptens was clustered on the distearoylphosphatidylcholine membranes, but with increase of cholesterol, random distribution of the haptens on the membranes increased. The cholesterol-dependent change in the topographical distribution of the haptens on the membranes paralleled the change of immunogenicity, i.e., the immunogenicity was low when haptens were clustered on the liposomal membranes. Haptens arranged at a proper distance on the membranes may be required for optimum immune response.     

Specific binding of H3-GABA by synaptic membranes of hypothalamus and hippocampus in rats after adrenalectomy and administration of hydrocortisone and corticosterone]

It has been shown that single hydrocortisone administration increased 3H-GABA binding by hypothalamic synaptic membranes. ACTH administration enhanced binding in both studied brain structures. Multiple hydrocortisone administration did not effect 3H-GABA binding by hypothalamic and hippocampal membranes, while multiple ACTH administration caused the decrease in mediator binding by hypothalamic membranes and increased its level in hippocampal membranes. Adrenalectomy did not change 3H-gaba binding and single hydrocortisone administration to adrenalectomized rats increased 3H-GABA binding only by hypothalamic synaptic membranes.     

Effect of polyunsaturated fatty acids and phospholipids on [3H]-vitamin E incorporation into pulmonary artery endothelial cell membranes

Vitamin E, a dietary antioxidant, is presumed to be incorporated into the lipid bilayer of biological membranes to an extent proportional to the amount of polyunsaturated fatty acids or phospholipids in the membrane. In the present study we evaluated the distribution of incorporated polyunsaturated fatty acids (PUFA) and phosphatidylethanolamine (PE) in various membranes of pulmonary artery endothelial cells. We also studied whether incorporation of PUFA or PE is responsible for increased incorporation of [3H]-vitamin E into the membranes of these cells. Following a 24-hr incubation with linoleic acid (18:2), 18:2 was increased by 6.9-, 9.2-, and 13.2-fold in plasma, mitochondrial, and microsomal membranes, respectively. Incorporation of 18:2 caused significant increases in the unsaturation indexes of mitochondrial and microsomal polyunsaturated fatty acyl chains (P less than .01 versus control in both membranes). Incubation with arachidonic acid (20:4) for 24 hr resulted in 1.5-, 2.3-, and 2.4-fold increases in 20:4 in plasma, mitochondrial, and microsomal membranes, respectively. The unsaturation indexes of polyunsaturated fatty acyl chains of mitochondrial and microsomal membranes also increased (P less than .01 versus control in both membranes). Although incubations with 18:2 or 20:4 resulted in several-fold increases in membrane 18:2 or 20:4 fatty acids, incorporation of [3H]-vitamin E into these membranes was similar to that in controls. Following a 24-hr incubation with PE, membrane PE content was significantly increased, and [3H]-vitamin E incorporation was also increased to a comparable degree, i.e., plasma membrane greater than mitochondria greater than microsomes. Endogenous vitamin E content of the cells was not altered because of increased incorporation of PE and [3H]-vitamin E. When [3H]-vitamin E was incorporated into lipid vesicles prepared from the total lipid extracts of endothelial cells and varying amounts of exogenous PE, vitamin E content was directly related to PE content. These results demonstrate that PUFA and PE distribute in all pulmonary artery endothelial cell membranes. However, only increases in PE were associated with increased incorporation of [3H]-vitamin E in membranes of these cells.     

The interactions of bilirubin with model and biological membranes

The partitioning of bilirubin between albumin and model and biological membranes and the differential partitioning of bilirubin between membranes with different lipid and protein compositions were measured. Partition coefficients were independent of the concentration of bilirubin in membranes up to at least 7 mol of bilirubin/mol of phospholipid. The avidity of albumin for bilirubin was greater than that of membranes, but the avidity of the latter for bilirubin depended on the composition of the membrane. Bilirubin partitioned preferentially into model membranes comprised of microsomal lipids greater than dioleoylphosphatidylcholine = plasma membrane lipids much greater than egg phosphatidylcholine = dimyristoylphosphatidylcholine. Partitioning into membranes was increased if these contained proteins, but the effect of proteins could not be attributed to specific binding to sites on proteins, as reflected by the temperature independence of partition coefficients. Differential partitioning of bilirubin into different membranes of pure lipids also was independent of temperature. Differences in the bulk phase fluidity of membranes does not appear to account for the preferential partitioning of bilirubin into some membranes. It appears that bilirubin partitions into elements of free volume of differing sizes in membranes with variable lipid compositions and that the size of these elements can be increased by adding proteins to membranes.     

Light-enhanced cross-linking of rhodopsin in rod outer segment membranes as detected by chemical probes

Bovine rod outer segment membranes were treated with cross-linking reagents before and after light exposure. Bleached membranes showed enhanced cross-linking with difluorodinitrobenzene or methyl acetimidate compared to dark-adapted membranes. The light-induced enhancement of cross-linking may be due to increased association of rhodopsin monomers in the light and/or due to increased reactivity of amino and sulfhydryl groups of bleached rhodopsin. In some instances, the band ascribed to the rhodopsin monomer in gel electrophoresis appears as a partially resolved doublet. Treatment of bleached rod outer segment membranes with methyl acetimidate improved the resolution of the doublet into two closely migrating bands.     

Enzymes that degrade phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate have different developmental profiles in chick brain

The activities and subcellular distributions of the hydrolases that degrade polyphosphoinositides were compared in the developing chick central nervous system. Specific activities increased 2- 3-fold and total activities increased 13- to 16-fold. Phosphatidylinositol 4-phosphate phosphatase is localized in membranes (78%), but is preferentially associated with nonmyelin membranes, since the increase in specific activity preceded myelination and proportions of membrane and soluble activities were constant during accumulation of myelin membranes. Phosphatidylinositol 4,5-bisphosphate phosphatase is largely soluble in embryonic (57%) and myelinated brain (50%). Although specific activity increased coincident with myelination, approximately equal increases in soluble and membrane activity indicate no preferential association with myelin membranes. Phosphatidylinositol 4,5-bisphosphate phosphodiesterase activity increased only in the early stages of myelination, but showed some preferential association with myelin membranes, since the proportion of soluble diesterase declined from 40 to 25%.     

The Effect of PS Content on the Ability of Natural Membranes to Fuse with Positively Charged Liposomes and Lipoplexes

Supramolecular aggregates containing cationic lipids have been widely used as transfection mediators due to their ability to interact with negatively charged DNA molecules and biological membranes. First steps of the process leading to transfection are partly electrostatic, partly hydrophobic interactions of liposomes/lipoplexes with cell and/or endosomal membrane. Negatively charged compounds of biological membranes, namely glycolipids, glycoproteins and phosphatidylserine (PS), are responsible for such events as adsorption, hemifusion, fusion, poration and destabilization of natural membranes upon contact with cationic liposomes/lipoplexes. The present communication describes the dependence of interaction of cationic liposomes with natural and artificial membranes on the negative charge of the target membrane, charges which in most cases were generated by charging the PS content or its exposure. The model for the target membranes were liposomes of variable content of PS or PG (phosphatidylglycerol) and erythrocyte membranes in which the PS and other anionic compound content/exposure was modified in several ways. Membranes of increased anionic phospholipid content displayed increased fusion with DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane) liposomes, while erythrocyte membranes partly depleted of glycocalix, its sialic acid, in particular, showed a decreased fusion ability. The role of the anionic component is also supported by the fact that erythrocyte membrane inside-out vesicles fused easily with cationic liposomes. The data obtained on erythrocyte ghosts of normal and disrupted asymmetry, in particular, those obtained in the presence of Ca²⁺, indicate the role of lipid flip-flop movement catalyzed by scramblase. The ATP-depletion of erythrocytes also induced an increased sensitivity to hemoglobin leakage upon interactions with DOTAP liposomes. Calcein leakage from anionic liposomes incubated with DOTAP liposomes was also dependent on surface charge of the target membranes. In all experiments with the asymmetric membranes the fusion level markedly increased with an increase of temperature, which supports the role of membrane lipid mobility. The decrease in positive charge by binding of plasmid DNA and the increase in ionic strength decreased the ability of DOTAP liposomes/lipoplexes to fuse with erythrocyte ghosts. Lower pH promotes fusion between erythrocyte ghosts and DOTAP liposomes and lipoplexes. The obtained results indicate that electrostatic interactions together with increased mobility of membrane lipids and susceptibility to form structures of negative curvature play a major role in the fusion of DOTAP liposomes with natural and artificial membranes.     

Subcellular proteomics of cell differentiation: quantitative analysis of the plasma membrane proteome of Caco-2 cells

Human colorectal carcinoma (Caco-2) cells undergo in culture spontaneous enterocytic differentiation, characterized by polarization and appearance of the functional apical brush border membrane. To provide insights into the biology of differentiation, we have performed a comparative proteomic analysis of the plasma membranes from proliferating cells (PCs) and the apical membranes from differentiated cells (DCs). Proteins were resolved by SDS-PAGE, in-gel digested and analyzed by RP-LC and MS/MS. Alternatively, proteins were digested in solution, and tryptic peptides were labeled with isotopic tags and analyzed by 2-D LC followed by MS/MS. Among the 1125 proteins identified in both proteomes, 76 were found to be significantly increased in the membranes of DCs and 61 were increased in PCs. Majority of the proteins increased in the apical membranes were metabolic enzymes, proteins involved in the maintenance of cellular structure, transmembrane transporters, and proteins regulating vesicular transport. In contrast, majority of the proteins increased in the membranes of PCs were involved in gene expression, protein synthesis, and folding. Both groups contained many novel proteins with yet to be identified functions, which could provide potential new markers of the intestinal cells or of colorectal cancer.     

Effects of alpha-tocopherol on the lipid peroxidation and fluidity of porcine intestinal brush-border membranes

The effect of alpha-tocopherol on the lipid fluidity of porcine intestinal brush-border membranes was studied using pyrene as a fluorescent probe. Addition of alpha-tocopherol to the medium decreased fluorescence intensity and lifetime, but increased the fluorescence polarization of pyrene-labeled membranes. beta-, gamma-, and delta-Tocopherols gave no appreciable effect on the fluorescence intensity and polarization of the complex. The apparent dissociation constant (3.1 +/- 0.12 microM) of the interaction of alpha-tocopherol with the membranes, estimated from the change in the fluorescence intensity with varying concentrations of alpha-tocopherol, was in good agreement with the concentration required to cause the half-maximal inhibition of lipid peroxidation of the membranes performed by incubation with 100 microM ascorbic acid and 10 microM Fe2+. Decrease of the slope in the thermal Perrin plot of the polarization of pyrene-labeled membranes by alpha-tocopherol suggests that the movement of pyrene molecules in the membranes is restricted by binding of the tocopherol. This interpretation was confirmed by an increased harmonic mean of the rotational relaxation time of the dye molecules in the membranes from 10.9 +/- 0.16 to 18.5 +/- 0.51 microseconds after addition of 25 microM alpha-tocopherol to the medium. The perturbation of lipid phase in the membranes induced by alpha-tocopherol was also suggested from a decreased quenching rate constant of pyrene fluorescence in the membranes for Tl+. Based on these results, the effect of alpha-tocopherol on the lipid fluidity of the membranes is discussed.     

Localization of a 5alpha-reductase activity in the nuclear membranes of the anterior hypophysis of normal and castrated adult male rats]

A 5alpha-reductase activity may be found in purified nuclei from anterior pituitary of intact or castrated male rats. When nuclear membranes are separated from nucleoplams this enzymic activity is recovered in nuclear membranes. Following 7 days of castration, the 5alpha-reductase activity in nuclear membranes is increased by 105 % with regard to the activity in nuclear membranes from intact animals.     

The mechanism of the stabilization of the hexagonal II (HII) phase in phosphatidylethanolamine membranes in the presence of low concentrations of dimethyl sulfoxide

Dimethyl sulfoxide (DMSO), a water-miscible organic solvent, has been used as a cryoprotectant for cells. It is known that DMSO stabilizes the HII phase of phosphatidylethanolamine (PE) membranes rather than the Lalpha phase, while most other water-miscible organic solvents such as acetone and ethanol destabilize the HII phase. To elucidate the mechanism for this stabilizing effect of DMSO on the HII phase, we have investigated its effects on the structures and physical properties of PE membranes. X-ray diffraction data indicated that dipalmitoleoylphosphatidylethanolamine (DPOPE) membranes in H2O at 20 degrees C were in the Lalpha phase and that an Lalpha to HII phase transition occurred at X=0.060 (mole fraction of DMSO) in water/DMSO mixtures. As the DMSO concentration increased, the basis vector length of the dioleoylphosphatidylethanolamine (DOPE)/ 16 wt% tetradecane membrane and also of the DPOPE/ 16 wt% tetradecane membrane in the HII phase decreased, suggesting that the spontaneous curvature of these membranes increased. We have also investigated the effects of DMSO on the physical properties of the PE membranes, and compared them with those of acetone. As the DMSO concentration increased, the excimer to monomer fluorescence intensities of pyrene-phosphatidylcholine in the PE membranes decreased, indicating that the membrane fluidity decreased, and also the generalized polarization value of the Laurdan fluorescent probe in the DPOPE membrane increased, indicating that the polarity of the membrane interface decreased. On the other hand, acetone had the opposite effects to DMSO. The interaction free energy between the membrane surface segments and solvent increased with an increase in DMSO concentration. It decreased the amount of solvent in the membrane interface, inducing an increase in the spontaneous curvature. This can reasonably explain the effects of DMSO on the phase stability and the physical properties of the membranes.     

Phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis as a probe for the distribution of phosphatidylinositol in hepatocyte membranes.

Phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Bacillus thuringiensis has been used as a probe for the distribution of phosphatidylinositol in hepatocyte membranes. Approx. 50% of this phospholipid was hydrolysed in microsomal vesicles (endoplasmic reticulum) with no significant hydrolysis of the remaining membrane phospholipids. Latency of mannose-6-phosphatase was retained during treatment indicating that the vesicles remained impermeable. Stripping of the ribosomes did not increase hydrolysis of phosphatidylinositol; however, when the vesicles were opened using dilute sodium carbonate, hydrolysis increased to greater than 90%. Hydrolysis of phosphatidylinositol of Golgi membranes was 35% and of plasma membranes was 50%. After treatment with PI-PLC, radiolabelled secretory proteins were retained in Golgi membranes and trapped lactate dehydrogenase was retained in plasma-membrane preparations indicating that the vesicles remained closed. Hydrolysis of phosphatidylinositol increased to greater than 90% when the membranes were opened by treatment with dilute sodium carbonate. These observations indicate that PI-PLC of Bacillus thuringiensis is a suitable probe for the distribution of phosphatidylinositol in membranes, and that in liver membranes this phospholipid occurs on each side of the bilayer, a topography consistent with its diverse roles.     

Membrane fluidity of platelets and erythrocytes in patients with Alzheimer's disease and the effect of small amounts of aluminium on platelet and erythrocyte membranes

The membrane fluidity of platelet and erythrocyte membranes in 10 Alzheimer's disease patients and 9 age-matched controls was studied. The platelet membranes of patients with Alzheimer's disease were found to be significantly more fluid than those of controls (p<0.02). However, erythrocyte membranes of Alzheimer patients were less fluid (more viscous) than those of controls (p<0.05). On further investigation of platelet and erythrocyte membranes obtained from healthy volunteers, the fluidity was found to change with increasing aluminium concentrations. When aluminium ammonium sulphate (0.01–10 M) was added to membrane suspensions, the fluidity of platelet membranes was increased, whereas the fluidity of erythrocyte membranes was decreased (i.e. the microviscosity was increased).     

Chronic and acute ethanol treatment modifies fluidity and composition in plasma membranes of a human hepaic cell line (WRL-68)

The aim of this study was to compare the effects of chronic (0.1 mol/L ethanol exposure during 30 days) and acute (0.5 mol/L ethanol exposure during 24 h) ethanol treatment on the physical properties and the lipid composition of plasma membranes of the WRL-68 cells (fetal human hepatic cell line). Using fluorescence polarization we found that ethanol treatment reduced membrane anisotropy due to disorganization of acyl chains in plasma membranes and consequently increased fluidity, as measured with the diphenylhexatriene probe. Addition of ethanolin vitro reduced anisotropy in control plasma membranes, whereas chronically ethanol-treated plasma membranes were relatively tolerant to thein vitro addition of ethanol. Acutely ethanol-treated plasma membranes exhibited a smaller anisotropy parameter value than control plasma membranes. We found a decrease in total phospholipid content in acute ethanol WRL-68 plasma membranes. Cholesterol content was increased in both ethanol treatments, and we also found a significant decrease in phosphatidylinositol and phosphatidylcholine and an increase in phosphatidylethanolamine content in ethanol-treated plasma membranes. Our data showed that ethanol treatment decreased the anisotropy parameter consistently with increased fluidity, while increasing the cholesterol/phospholipid ratio of plasma membranes of WRL-68 cells, but only chronically ethanol-treated plasma membranes exhibited tolerance to thein vitro addition of ethanol. It is important to note that some changes that were interpreted as a result of chronic ethanol treatment were also present in short-period ethanol treatments.Abbreviations DPH diphenylhexatriene - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SPH sphingomyelin     

Development of parthenogenetic membranes in double-yolked and injected chicken eggs.

Development of parthenogenetic eggs is expressed in a Dark Cornish stock by either membrane or embryo formation. Double-yolked eggs have membranes developing in zero, one, or two yolks. The incidence of double-yolked eggs with two membranes was higher than expected when compared with the incidence of single-yolked eggs from the same hens. Membrane formation was increased over that of the controls by injecting saline, Ringer's solution, or ground crude membranes. Yolk and membranes filtered through a 3 mu filter or autoclaved did not increase membrane formation over that of the controls. Fertile membranes behaved like parthenogenetic membranes.     

Effects of acute and chronic endotoxin treatment on glucagon and insulin receptors on rat liver plasma membranes

The effects of acute and chronic endotoxin treatment on the plasma levels of insulin and glucagon and their binding to rat liver plasma membranes were examined. Both acute and chronic endotoxin administration increased plasma glucagon levels and decreased the glucagon to insulin molar ratio. Acute, but not chronic, endotoxin decreased blood glucose and insulin levels. Glucagon binding was increased in membranes prepared from the acutely treated rats. However, in membranes obtained from rats treated chronically with endotoxin, only insulin binding was increased. The increases in the binding of both insulin and glucagon were the result of increases in receptor sites.     

Effect of aging on the composition of mitochondrial membranes from potato slices

The phospholipid content of mitochondrial membranes from slices of potato tuber (Solanum tuberosum) remains stable during aging. The phospholipid compositions of whole mitochondria and inner membranes do not vary during aging whereas the concentrations of phosphatidylinositol and phosphatidyl-glycerol in outer membranes are slightly amplified. The saturation of outer membrane fatty acids is slightly increased during aging. Gel electrophoresis of mitochondrial membrane proteins show slight variations of one polypeptide in outer membranes and of three polypeptides in inner membranes. These results suggest parallel variations of lipids and proteins in membranes during aging, in marked contrast with the large modifications observed in mitochondrial activities.     

Effects of 2-hydroxyoleic acid on the structural properties of biological and model plasma membranes

Genetic hypertension is associated with alterations in lipid metabolism, membrane lipid composition and membrane-protein function. 2-Hydroxyoleic acid (2OHOA) is a new antihypertensive molecule that regulates the structure of model membranes and their interaction with certain peripheral signalling proteins in vitro. While the effect of 2OHOA on elevated blood pressure is thought to arise through its influence on signalling proteins, its effects on membrane lipid composition remain to be assessed. 2OHOA administration altered the lipid membrane composition of hypertensive and normotensive rat plasma membranes, and increased the fluidity of reconstituted liver membranes from hypertensive rats. In spontaneously hypertensive rats (SHR), treatment with 2OHOA increased the cholesterol and sphingomyelin content while decreasing that of phosphatidylserine-phosphatidylinositol lipids. In addition, monounsaturated fatty acid levels increased as well as the propensity of reconstituted membranes to form HII-phases. These data suggest that 2OHOA regulates lipid metabolism that is altered in hypertensive animals, and that it affects the structural properties of liver plasma membranes in SHR. These changes in the structural properties of the plasma membrane may modulate the activity of signalling proteins that associate with the cell membrane such as the Galphaq/11 protein and hence, signal transduction.     

Liver ischemia increases the molecular order of microsomal membranes by increasing the cholesterol-to-phospholipid ratio

An accelerated degradation of phospholipid is the likely basis of irreversible cell injury in ischemia, and the membranes of the endoplasmic reticulum of the liver are a convenient system with which to study the effect of such a disturbance on the structure and function of cellular membranes. In the present report, electron spin resonance spectroscopy has been used to evaluate changes in the molecular ordering of microsomal membrane phospholipids in the attempt to relate the loss of lipid to alterations in membrane structure. The order parameter, S, was calculated from spectra reflecting the anisotropic motion of 12-doxyl stearic acid incorporated into normal and 3-h ischemic microsomal membranes. Over the temperature range 4-40 degrees C, the molecular order (S) of ischemic membranes was increased by 8-10%. This increase was reproduced in the ordering of the phospholipids in liposomes prepared from total lipid extracts of the same membranes. In contrast, after removal of the neutral lipids, liposomes prepared from phospholipids of ischemic and control membranes had the same molecular order. There were no differences in the phospholipid species of control and ischemic membranes or in the fatty acid composition of the phospholipids. In the neutral lipid fraction of ischemic membranes, however, triglycerides and cholesterol were increased compared to control preparations. There were no free fatty acids. The total cholesterol content of the liver was unchanged after 3 h of ischemia. The cholesterol-to-phospholipid ratio of ischemic membranes, however, was increased by 22% from 0.258 to 0.315 as a consequence of the loss of phospholipid. Addition of cholesterol to the control total lipid extracts to give a cholesterol-to-phospholipid ratio the same as in ischemic membranes resulted in liposomes with order parameters similar to those of liposomes prepared from ischemic total lipids. It is concluded that the degradation of the phospholipids of the microsomal membrane results in a relative increase in the cholesterol-to-phospholipid ratio. This is accompanied, in turn, by an increased molecular order of the residual membrane phospholipids.     

Erythrocyte membrane deformability and stability: two distinct membrane properties that are independently regulated by skeletal protein associations

Skeletal proteins play an important role in determining erythrocyte membrane biophysical properties. To study whether membrane deformability and stability are regulated by the same or different skeletal protein interactions, we measured these two properties, by means of ektacytometry, in biochemically perturbed normal membranes and in membranes from individuals with known erythrocyte abnormalities. Treatment with 2,3-diphosphoglycerate resulted in membranes with decreased deformability and decreased stability, whereas treatment with diamide produced decreased deformability but increased stability. N-ethylmaleimide induced time-dependent changes in membrane stability. Over the first minute, the stability increased; but with continued incubation, the membranes became less stable than control. Meanwhile, the deformability of these membranes decreased with no time dependence. Biophysical measurements were also carried out on pathologic erythrocytes. Membranes from an individual with hereditary spherocytosis and a defined abnormality in spectrin-protein 4.1 association showed decreased stability but normal deformability. In a family with hereditary elliptocytosis and an abnormality in spectrin self-association, the membranes had decreased deformability and stability. Finally, membranes from several individuals with Malaysian ovalocytosis had decreased deformability but increased stability. Our data from both pathologic membranes and biochemically perturbed membranes show that deformability and stability change with no fixed relationship to one another. These findings imply that different skeletal protein interactions regulate membrane deformability and stability. In light of these data, we propose a model of the role of skeletal protein interactions in deformability and stability.